Improvements have been made in retroviral mediated transduction of fetal tissues. However, in utero gene therapy, like other gene therapy applications, has been hampered by severely limited levels of transgene expression and a lack of persistent expression in differentiated cells due to promoter silencing mechanisms. Although some genetic therapy applications for genetic diseases of the hematopoietic system may not require cell specific transgene expression, for others targeted expression may be an essential prerequisite for treatment of these disorders. The long-term goal of the proposed research is the development novel retroyiral vectors which direct high level, persistent, and cell specific transgene expression in differentiated graniiloc and monocytes for use in our ongoing in utero gene therapy studies. We propose to investigate promoter regions from myeloid specific genes C/EBPepsilon and CD 11b to direct cell type specific transgene expression. To overcome the problem of low and transient transgene expression levels we propose to augment our cell specific expression vectors with tandem repeats of a myeloid enhancer region which may enhance promoter activity and/or chromatin insulators to eliminate chromatin silencin effects. These expression vectors will be investigated for their ability to direct high, protracted, myeloid specific transgene expression by transduction into human myeloid cell lines and primary human CD34+ cells followed by in vitro differentiation. Transplantation of transduced human CD 34+ cells into irradiated SCID mice to recapitulate human hematopoiesis and subsequent analysis of gene expression in differentiated hematopoietic cells will be performed. Finally, the vector will analyzed for their ability to direct high, protracted, myeloid specific transgene expression by injection of retroviral supernatant into fetal mice. The results of these studies will provide critical information regarding the feasibility of persistent, cell specific gene expression for use in the in utero treatment of inherited hematopoietic diseases.